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High Sensitivity Tetraspan Tracking (HSTT) ELISA Kit

Research Use Only Not for Diagnostic Use

Description: EVix High Sensitivity Tetraspan Tracking (HSTT) kit quantifies tetraspan (CD9, CD63, and CD81) antigen in an EV sample.  EV are captured in individual wells coated with anti CD9, CD63 and CD81 monoclonal antibodies.  The tri-antibody coating maximizes capture of EVs expressing any or all of the three tetraspan antigens.

The high affinity and specificity of the capture provides sufficient capture in two hours of incubation.  The detection antibody cocktail is also highly sensitive allowing quantification of EV preparations based on antigen content. In contrast to NTA analysis which can be time consuming to run multiple samples, the HSTT assay can quantitate antigen content of an numerous EV preparation in less than 4 hrs total. The assay is straightforward, sensitve and reproducible.

The HSTT assay kit includes an internal standard that yields a highly reproducible signal and can be used to calibrate EV preparations.  This allows dilution or concentration of EV preparations to achieve equivalency for downstream investigation.  The HSTT assay can be used to assess multiple EV samples from the same source or to compare EV preparations from different sources.

The assay kit comes complete with all reagents needed to obtain results.  No other reagents are needed to obtain values for EV samples.  The HSTT assay is the answer to quantifying EV preparations.  Simple, rapid, highly sensitive and reproducible.  Start measuring your EV preparations based on non destructive antigen capture and recognition.  Track your purification strategies, calibrate preparations, compare recovery rates, and more.

HSTT Gauge 2

HSTT Values for Tracking Tetraspan Content

Use our HSTT Assay to monitor tetraspan antigen content in your EV preparations.  High sensitivity and specificity for capture and detection is assured with anti tetraspan monoclonal antibodies.   Create numerical values for EV preparations to equilibrate or normalize EV preparations.

HSTT values are used to guide dilution or concentration of preparations for down stream analysis.  These values allow for normalizing samples to be assayed in the Multi-Tope and Single-Tope assays.  Samples diluted to equivalent HSTT values can be run in Multi-Tope and Single-Tope assays and compared for antigen distribution patterns.

Complement your NTA analysis with HSTT values.  Once your purification process is locked in, HSTT can substitute for the laborious NTA analysis to quantify EV preparations.  Calibrating your NTA values to HSTT readings may allow you to substitute HSTT to follow EV containing antigen amounts in your experiments.


HSTT Dilution Curve

Sensitive and Reproducible

Replicate measurements of an EV preparation show the reproducibility of HSTT values.   Three different assays ran on the same sample.  Values above 3.5 AU are close to saturation.  EV preparations were diluted in wash buffer by serial dilution.  EV preparations were from SW 620 culture supernatants diafiltered against 300,000 MWCO membrane concentrator.

HSTT Standrd Curve

Quantify EV Preparations

Linearity of HSTT assay values to increased antigen amounts is shown on left.  Three different assays of the same EV preparation demonstrate the sensitivity and linearity of assay values to increasing amounts of EVs.   Values above 3.5 AU are close to saturation.  EV preparations were diluted in wash buffer by serial dilution.  EV preparation was from SW 620 culture supernatant, diafiltered against 300,000 MWCO concentrator membrane.  A 1 to 10 dilution of supernatant yielded near maximum assay values.

HSTT Samples

Normalize Different EV Preparations

EV Preparations from 10 different biological sources were assayed at 4 dilutions to compare HSTT values.  Each preparation resulted in a different HSTT dilution curve.  Samples included normal urine, Mixed lymphocyte reaction (MLR) supernatants, and indicated cell line supernatants.  All samples were dia-filtered against a 300,000 MWCO membrane.  The EV content of the individual samples can be normalized to equivalent HSTT values by either concentration or dilution.  It is recommended that a HSTT value greater than 3.0 be obtained for an EV sample prior to Multi-Tope and Single-Tope assay.


Dilute or Concentrate EV preparations to equivalent HSTT values

Dilution of EV Preparations from 10 different biological sources reveal unique HSTT dilution values for each preparation.  Dilution from concentrate for all samples are shown. Samples included normal urine, MLR supernatants, and cell line supernatants.  All samples were dia-filtered against a 300,000 MWCO membrane.



HSTT Serum

High Sensitivity

Nomal human serum was ran on a S300 HR Sephacryl chromatography column.  Fractions eluted off of the column were than analyzed for HSTT activity.  0.5 ml of serum was loaded onto column and 0.5 ml fractions collected.  The 260 and 280 absorbance values for the fractions are shown on the left vertical axis.  HSTT values of the individual fractions are shown on the right vertical axis.  Note robust HSTT signals in the exclusion/void volume but not in the inclusion volume.  IgM also came out in void volume.

HSTT signals were readily detected in the fractions even when the serum sample was diluted several fold due to fraction collection.

  • Antigen Capture Targeting Human Tetraspans

    Capture EVs with high specificity monoclonal antibodies targeting CD9, CD63 and CD81

  • Detect Captured EVs Targeting Human Tetraspans

    Detect captured tetraspan containing EVs with a triple cocktail of anti CD9, CD63 and CD81 biotinylated antibodies.

  • High Sensitivity

    EV detection in culture supernatants and serum with out concentration

  • Efficient

    Run multiple samples simultaneously, run dilutions in less time than it would take to run an NTA analysis.

  • Standardize EV Preparations

    Quantify EV tetraspan antigen content.  Use values to normalize different EV preparations for downstream analysis and phenotyping.

  • Reproducible

    HSTT values can be compared from assay to assay.  The included standard provides a means to assess results between assays. 

  • Calibrate EV Preparation for Multi-Tope Data

    The Multi-Tope assay reveals additional antigens associated with the tetraspans CD9, CD63 and CD81.  Values from the HSTT assay are used to normalize EV preparations by tetraspan signal prior to Multi-Tope assay. 

Kit Contents:  

A: EVix Capture Plate:  (12 x 8 well strips) and frame.  

B: 10X Sample and Wash Buffer Concentrate: 2 bottles (10 ml each) to make 200 ml. 

C: Biotinylated Primary Detection Triple Antibody Cocktail : 1 vial of 0.5 ml to make 10 ml.

D:  Streptavidin Horse Radish Peroxidase SAHRP Conjugated: 1 vial of 0.5 ml to make 10 ml. 

E: Chromogenic Solutions : 1 bottle (10 ml) of  stabilized H2O2 , 1 vial (0.5 ml) of stabilized TMB chromogen.  Caution!  

F: Stop Solution: 1 bottle (10 ml) Acid Caution! 

G: EVTetraspan Standard: 1 vial 0.5 ml.

If contents are unopened and stored at 4o C the kit is stable for 6 months.

  • Order HSTT-001.  
  • 96 wells in 8 well modular strips.  
  • All assay components included.

Catalog HSTT-001

96 Wells